The use of the biguanide drug, metformin, is standard for young women with type 2 diabetes and is becoming prevalent for the treatment of hyperandrogenism and anovulation in women with polycystic ovary syndrome (PCOS). In vitro studies have shown that metformin has direct effects on inhibiting ovarian thecal androgen biosynthesis, which is of clinical relevance to both groups of women. However, the mechanism(s) by which metformin inhibits ovarian androgen biosynthesis is presently unclear. Our preliminary studies suggest that metformin treatment has differential effects on inhibiting androgen biosynthesis in theca cells propagated from normal cycling and PCOS women. An examination of the classical metformin activated LKB1/ AMP-activated protein kinase (AMPK) signaling cascade, provided the novel findings that LKB1 phosphorylation, as well as AMPK activity are decreased in PCOS theca cells. Metformin treatment corrected these signaling defects in PCOS theca cells, while inhibiting androgen biosynthesis. In this proposal we will test the hypothesis that defects in LKB1/AMPK signaling in PCOS theca cells contribute to increased androgen biosynthesis and disrupted cell growth in the PCOS ovary. In Specific Aim 1, we will investigate the underlying mechanism(s) by which metformin activates the LKB1/AMPK signaling pathway(s) to inhibit androgen biosynthesis in PCOS theca cells. We will examine the role(s) of LKB1, AMPK, and the salt inducible kinases, SIK1 and SIK2, in metformin inhibition of CYP17 and CYP11A1 gene expression and androgen biosynthesis in normal and PCOS theca cells propagated in long-term culture. The pathological characteristic of the PCOS ovary is that of multiple, small, growth arrested follicles. However, there have been no reports comparing cell growth or cell cycle control in normal and PCOS theca cells. We have identified differences in cell cycle regulation in normal and PCOS theca cells, and observed that metformin treatment of PCOS theca cells was observed to change PCOS and cell cycle progression to a normal phenotype. In Specific Aim 2, we will identify cell growth and cell cycle defects in PCOS theca cells and explore the signaling mechanism(s) involved in metformin's ability to correct these defects. The roles of LKB1, AMPK, and SIK1/2 in metformin-dependent cell growth and cell cycle progression, under normal physiological and hyperinsulinemic conditions, will be evaluated. We believe a clear understanding of mechanism(s) by which metformin signals to mediate inhibition of androgen biosynthesis and modulate cell growth in PCOS theca cells will help elucidate the pathophysiology of PCOS, and provide insight for the development of new therapeutic targets for PCOS. PUBLIC HEALTH RELEVANCE: Polycystic ovary syndrome (PCOS) is characterized by ovarian cysts, anovulation, hyperandrogenism, and endometrial hyperplasia, and affects 6-10% of reproductive age women, approximately 3-5 million women in the United States alone. Hyperandrogenism and anovulation in women with PCOS can be clinically treated with administration of the insulin sensitizing drug, metformin, a treatment for type 2 diabetes, however the mechanisms by which metformin directly modulates the ovary have not been examined. A rigorous evaluation of the mechanism(s) by which the metformin activates the LKB1/AMPK signaling pathway(s) to correct defects in androgen biosynthesis and cell growth in PCOS theca cells, will provide essential insight for the development of new therapeutic modalities for PCOS.